Analysis of circulating cell-free DNA (cfDNA) in plasma has several established and upcoming diagnostic applications in prenatal, cancer and transplant medicine (1-7). Whole-genome sequencing of cfDNA is clinically available for noninvasive prenatal screening of fetal aneuploidies (1, 5). Next-generation sequencing of cfDNA in cancer patients can enable noninvasive identification of cancer mutations, monitoring of cancer burden and tumor evolution (4, 8-10). However, little is understood about the effect of pre-analytical factors on DNA quality and on performance of molecular assays.
Informative fraction of cfDNA is generally fragmented with a modal size of 160-180 bp. Pre-analytical factors such as delayed fractionation of plasma or incomplete removal of peripheral blood cells before freezing can cause an increase in higher molecular weight (HMW) DNA fraction from cell lysis. For PCR-based sequencing approaches, this can artificially lower the fraction of the target alleles such as a tumor-specific mutations in circulating tumor DNA making its detection more challenging and causing errors in its quantification. Since cfDNA is fragmented in vivo, ligation-mediated preparation of sequencing libraries from cfDNA does not involve shearing, and therefore HMW DNA is not incorporated into sequencing libraries. If the upfront measurement of total cfDNA is erroneously high due to a large fraction of HMW DNA downstream sequencing can be compromised.
Quantification of cfDNA can be performed using fluorometric or spectrophotometric methods such as QUBIT® (Life Technologies) or NANODROP™ (Thermo Scientific). These methods do not measure DNA size and cannot account for a HMW fraction in plasma DNA. Electrophoretic methods can perform size-based quantification but require input amounts for reliable results that are not feasible for cfDNA analysis. In addition, none of these methods provide an assessment of amplifiable DNA copies available for downstream molecular analysis. Multiplexed quantitative PCR can provide an assessment of size and amplifiable DNA but requires comparison with a standard curve. It relies on 1-2 genomic loci to infer total cfDNA content assuming that the targeted loci are single-copy genes. This assumption can affect measurement of cfDNA from cancer patients with somatic copy number changes reflected in plasma. In addition, it was recently shown that relative readouts from multiple single-locus assays can vary systematically potentially due to assay performance and variable stability across genomic DNA (11)
An efficient method for accurate quantification of cfDNA and assessment of cfDNA integrity is needed. As the amounts of cfDNA available in a sample are generally very limited, such a method requires a relatively wide dynamic range and the capability to detect and assess minute amounts of cfDNA.